Journal
THERIOGENOLOGY
Volume 83, Issue 3, Pages 415-420Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.theriogenology.2014.10.006
Keywords
Preimplantation genetic diagnosis; Equine; Preimplantation embryo; Sex determination; Apoptosis
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Funding
- Instituto de Investigaciones Biotecnologicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martin
- Cria Tanoira Embriones
- Catedra de Teriogenologia, Instituto de Investigacion y Tecnologia en Reproduccion Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires
- Laboratorio de Produccion Equina, Universidad Nacional de Rio Cuarto
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Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo- and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR. (C) 2015 Elsevier Inc. All rights reserved.
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